Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain.
We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression.
Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

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"Membrane traffic pathways have also proven important for many processes in development, including the release of key developmental signals, the down-regulation of signaling receptors, and the establishment of both apical-basal and planar polarity (Baron, 2012; Dunst et al., 2015; Fürthauer and González-Gaitán, 2009; Lu and Bilder, 2005; Zacharogianni and Rabouille, 2013; Zhang et al., 2007). Finally, there is a growing interest in understanding how the basic machinery of membrane traffic is varied and augmented in the diverse cell types of a whole animal (Burgess et al., 2011; Chan et al., 2011; Fox et al., 2010; Giansanti et al., 2007; Lerner et al., 2013). Indeed, many cells differ greatly in subcellular organization and secretory function, and the Drosophila system provides an excellent model to understand how this diversity is established. "

[Show abstract] [Hide abstract] ABSTRACT: The use of Drosophila melanogaster as a model organism has been pivotal to understanding the developmental processes of metazoans. However, the use of flies for studying subcellular organization is hampered by a paucity of reliable reagents to label specific organelles. Here, we describe the generation of mouse monoclonal antibodies against a set of markers of the secretory and endocytic pathways, along with goat polyclonal antibodies against two Golgi proteins. We show that the monoclonal antibodies are highly specific and sufficiently sensitive to detect endogenous proteins in crude extracts by immunoblotting with little background staining. By immunofluorescence the major compartments of the membrane traffic system (including the endoplasmic reticulum, the Golgi, and early and late endosomes) are labeled by at least one antibody. Moreover, the antibodies can be used to label organelles in fly tissues including salivary glands and wing imaginal discs. We anticipate that these antibodies will provide a useful tool kit to facilitate the investigation of how the endomembrane system functions and varies in the diverse tissue types of metazoans.

"This emphasizes that endocytic delivery to the MVBs is required for loading Hh onto exovesicles rather than a requirement of rerouting it to other membrane domains for exovesicular secretion (D'Angelo et al., 2015). However, dRab27 is absent in the Drosophila wing imaginal disc (Chan et al., 2011), and hence we could not test the effects of this protein in the animal. There is a possibility of another Rab protein that could take over the MVB fusion function in the wing imaginal disc, which needs to be identified. "

[Show abstract] [Hide abstract] ABSTRACT: Hedgehog (Hh) is a secreted morphogen, involved in both short and long range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We had previously shown that the long range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multi vesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Importantly oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long range signaling.

"The Drosophila genome predicts 33 Rab proteins based on sequence similarity, and there is evidence that 27 are expressed (Chan et al., 2011; Zhang et al., 2007). Fourteen of these Rabs were already present in the LECA (between 2–3 billion years ago), and the other 13 first arose by duplication and divergence at the root of the metazoan lineage (500 million years ago). "

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